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Mucosal vaccine adjuvants containing bacterial flagellins as an active component

a technology of flagellin and mucosal vaccine, which is applied in the direction of antibody medical ingredients, drug compositions, instruments, etc., can solve the problems of reducing immunogenicity, raising a significant social problem, and insufficient human use, so as to stimulate the production of interleukin-8 and increase the mucosal iga

Active Publication Date: 2011-03-29
RHEE JOON HAENG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The use of flagellins as adjuvants significantly increases mucosal IgA production and provides complete protection against tetanus toxoid, demonstrating their effectiveness in inducing robust immune responses and serving as a safer alternative to existing adjuvants.

Problems solved by technology

Although the absolute number of clinical cases of this disease is less than that of cholera or salmonella food poisoning, it raises a significant social problem due to its high mortality rate and tragic clinical manifestations.
However the administration of protein antigens via the mucosal route has a disadvantage that immunogenicity is decreased compared to the administration via the systemic route.
However these adjuvants are exotoxins with high enterotoxicity, thus being inadequate to be used directly for human beings.

Method used

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  • Mucosal vaccine adjuvants containing bacterial flagellins as an active component
  • Mucosal vaccine adjuvants containing bacterial flagellins as an active component
  • Mucosal vaccine adjuvants containing bacterial flagellins as an active component

Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of Transposon Libraries

[0046]V. vulnificus MO6-24 / O type strains (obtained from J. Glenn Morris, Division of Hospital Epidemiology, University of Maryland School of Medicine, USA) and mini-Tn5 lacZ1 containing E. coli SM10λpir strains (obtained from Kenneth N. Timmis, GBF National Research Center for Biotechnology, Braunschweig, Germany) were cultured overnight at 37° C., 210 rpm in a stirring incubator, each were inoculated with single colony at 10 ml of 2.5 HI(2.5% NaCl heart infusion) broth media and 20 ml of LB (containing 100 μg / ml of Ampicillin and 100 μg / ml of Kanamicin) broth media

[0047]The following day these were centrifuged, and washed with antibiotic-free LB broth media and centrifuged two times, then suspended at 100 μl of new LB broth media. Each bacterial suspension of E. coli and V. vulnificus were mixted together and dropped on LB agar plate. After culturing it overnight at 37° C., 800 μl of new 2.5 HI broth media was added to the grown colonies on LB a...

example 2

Screening of Transposon Mutant Clones that Lose otility.

[0049]Each clone, prepared in Example 1, of the V. vulnificus MO6-24 / O transposon libraries was cultured overnight at 37° C., then inoculated to 0.3% agar containing semi-solid state HI (heart infusion) agar plates using sterilized toothpicks and cultured at 37° C. for 6 hours. Degrees of the motility of the bacteria were then determined by measuring the range of movement after growing the bacteria

[0050]3 transposon mutant clones that nearly completely lost motility were selected by screening procedures, and the experiment that would identify the mutant genes which insert transposons was progressed.

example 3

Identification of Flagellin Operon Genes

[0051]The cloning of genes nearby the transposon inserted region was carried out by screening the cosmid gene libraries, using DNA fragment as primer for amplification by arbitrary PCR methods. The amplification of DNA fragments nearby the transposon inserted site was used a two-step PCR amplification method. In the first PCR, the arbitrary primer 1 (5-GGCCACGCGTCGACTAGTCANNNNNNNNNNACGCCC-3) of sequence number 13, and the mini-Tn5 lacZ1 specific primer 1 (5-TTCTTCACGAGGCAGACCTCAGCGC-3) of sequence number 14, were used. The first PCR was set as follows; denaturing them for 30 seconds (sec) at 94° C., annealing for 30 sec at 30° C., and elongating for 1 minute (min) 30 sec at 72° C., with 5 cycles; afterwords a further 30 cycle PCR reaction was performed by denaturing for 30 seconds (sec) at 94° C., and annealing for 30 sec at 45° C., and elongating for 2 min at 72° C., with 30 cycles. The second PCR reaction was peformed using the products of t...

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Abstract

The present invention relates to mucosal vaccine adjuvants containing flagellins, the structural component of flagella, originated from Vibrio vulnificus, Salmonella typhimurium, and Listeria monocytogenes as an active component.

Description

TECHNICAL FIELD[0001]The present invention relates to mucosal vaccine adjuvants containing flagellins, the structural component of bacterial flagella, originated from Vibrio vulnificus, Salmonella typhimurium, and Listeria monocytogenes as an active component.BACKGROUND ART[0002]The infectious disease from Vibrio vulnificus or its abbreviation “V. vulnificus” has a relatively short history, but clinical cases have been reported continuously worldwide and this disease is one of the newly obserbed diseases. Although the absolute number of clinical cases of this disease is less than that of cholera or salmonella food poisoning, it raises a significant social problem due to its high mortality rate and tragic clinical manifestations.[0003]V. vulnificus was first reported in 1976 by Hollis et al. of CDC (Centers for Disease Control in USA) after they studied bacteriological properties of halophilic, pathogenic Vibrio that was isolated from human for 11 years, and named lactose-fermenting ...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): A61K45/00A61K39/02C07K14/00C07K1/00C07K17/00A61K39/08A61K39/39
CPCA61K39/08A61K39/39A61K2039/541A61K2039/55516A61K2039/55544A61P15/16A61P31/00A61P35/00Y02A50/30G02F1/1309G02F2203/69
Inventor RHEE, JOON-HAENGLEE, SHEE-EUNKIM, SOO-YOUNG
Owner RHEE JOON HAENG
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